We have identified Rho-Kinase (ROCK) as a novel regulator of insulin action and glucose homeostasis. Specifically, ROCK directly regulates IRS-1 function by phosphorylating serine residues. Inhibition of ROCK function results in a reduction of insulin-stimulated glucose transport and insulin signaling, leading to insulin resistance. We found that ROCK1 deficiency causes systemic insulin resistance in vivo by impairing insulin signaling in skeletal muscle. Furthermore, insulin-stimulated ROCK activity is decreased in the skeletal muscle of obese diabetic mice and humans, suggesting that defective ROCK activity may contribute to the pathogenesis of insulin resistance. We thus hypothesize that activation of ROCK1 is critical for the metabolic action of insulin on glucose transport and insulin signaling through IRS-1 serine phosphorylation. The overall objective of this proposal is to define the novel role of ROCK1 in the regulation of glucose metabolism and insulin signaling. Aim 1 works to determine the role of IRS-1 serine phosphorylation in ROCK1-dependent glucose metabolism. We will systemically investigate how ROCK1 controls insulin- mediated glucose metabolism via affecting IRS-1 serine phosphorylation. This will be achieved by reintroducing IRS-1 mutants in IRS-1-deficient cells and mice. Aim 2 further elucidates the molecular mechanism in which ROCK1 is a key regulator of insulin action on glucose transport and downstream signaling. We will use small RNA interference and adenovirus gene-transfer strategies to determine the fundamental basis underlying insulin-induced activation of ROCK1 in insulin-sensitive cells or specific cell lines in which ROCK1 or IRS-1 has been deleted. Aim 3 defines the role of ROCK1 in whole-body glucose homeostasis and insulin sensitivity in insulin-target tissues. We have successfully generated loxP flanked ROCK1 mice, which are currently being breed with mice expressing MCK-Cre or adiponectin-Cre recombinase producing muscle- or adipose-specific ROCK1-deficient mice. These mice will be characterized for whole-body insulin sensitivity and glucose homeostasis, with important implications for strategies to prevent diabetes. These studies provide a unique opportunity to identify a novel molecular mechanism in which ROCK1 is important as a critical node in insulin control of metabolism, and may offer a novel target for the treatment of diabetes and obesity.